In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartmentspecific measurements of intracellular pH (pH i ) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pH cyt ) and mitochondrial pH (pH mit ) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pH cyt of 7.2 and a constant pH mit of 7.5 in cells exponentially growing on glucose. We show that pH cyt and pH mit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pH i decrease coincided with growth stasis. Additionally, pH i and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pH i and cellular energy generation, and therefore a relation between pH i and growth. INTRODUCTIONMicrobes are able to adapt to a wide range of stressful environments such as deviant temperature, high or low osmotic pressure, oxidative stress and exposure to weak organic acids. The mechanisms by which they adapt to these environments are often poorly understood. To study these adaptive responses we rely on techniques that focus on various levels of cellular regulation, such as transcription profiles, protein levels and metabolic flux analysis. However, global physiological parameters such as intracellular pH (pH i ) affect nearly all processes in a living cell. pH i directly influences the redox state of the cell by influencing the NAD + /NADH equilibrium (Veine et al., 1998) and determines pH gradients necessary for transport over membranes (Goffeau & Slayman, 1981;Wohlrab & Flowers, 1982). Additionally, the effect of pH is very prominent in enzyme kinetics, as pH influences ionization states of acidic or basic amino acid side-chains and thereby influences the structure, solubility and activity of most, if not all, enzymes.The different organelles in the cell all maintain their own specific pH, which is used to define and maintain the processes associated with each organelle. Yeast vacuoles, for instance, are reported to have an acidic pH (Preston et al., 1989;Brett et al., 2005; Martínez-Muñoz & Kane, 2008). The proton gradient across the vacuolar membrane has been shown to be essential for the transport of various compounds (Nishimura et al., 1998;Ohsumi & Anraku, ...

Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

No abstract

BackgroundBecause protonation affects the properties of almost all molecules in cells, cytosolic pH (pHc) is usually assumed to be constant. In the model organism yeast, however, pHc changes in response to the presence of nutrients and varies during growth. Since small changes in pHc can lead to major changes in metabolism, signal transduction, and phenotype, we decided to analyze pHc control.ResultsIntroducing a pH-sensitive reporter protein into the yeast deletion collection allowed quantitative genome-wide analysis of pHc in live, growing yeast cultures. pHc is robust towards gene deletion; no single gene mutation led to a pHc of more than 0.3 units lower than that of wild type. Correct pHc control required not only vacuolar proton pumps, but also strongly relied on mitochondrial function. Additionally, we identified a striking relationship between pHc and growth rate. Careful dissection of cause and consequence revealed that pHc quantitatively controls growth rate. Detailed analysis of the genetic basis of this control revealed that the adequate signaling of pHc depended on inositol polyphosphates, a set of relatively unknown signaling molecules with exquisitely pH sensitive properties.ConclusionsWhile pHc is a very dynamic parameter in the normal life of yeast, genetically it is a tightly controlled cellular parameter. The coupling of pHc to growth rate is even more robust to genetic alteration. Changes in pHc control cell division rate in yeast, possibly as a signal. Such a signaling role of pHc is probable, and may be central in development and tumorigenesis.

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme-kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme-kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113-7D grown in aerobic glucose-limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h(-1). The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo-like medium containing 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM free magnesium and 0.5 mM calcium, at a pH of 6.8. The V(max) values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme-specific, optimal conditions.